Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys–/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (Em, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3–) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3–). The active site heme in the stable sulfonated protein displays an Em approximately 200 mV more positive than the Cys–/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

Protein purification: C. jejuni (Cj) TsdA was expressed in E. coli and purified as described previously. 1 The corresponding gene encodes the 309 amino acids predicted from C8J_0815 without the signal peptide, preceded by 15 N-terminal amino acids that include the Strep-II tag to facilitate purification. Residues are numbered from the first amino acid of this N-terminal extension. Sample preparation: Samples were prepared in aqueous 50 mM HEPES, 50 mM NaCl, pH 7 buffer (with the exception of those used for nIR MCD measurement where D2O replaced H2O 2 ) and transferred to an anaerobic chamber for incubation with anaerobic TCEP (final concentration, 1.5 mM), sulfite (final concentration, 1.5 mM) and/or ferricyanide (to achieve full re-oxidation) as indicated, Table S1. Samples (30 -50 μM protein) for LC-MS were additionally treated with iodoacetate to acetylate free (per)thiols unless indicated otherwise. Excess sulfite or ferricyanide was then removed either by passage down a PD-10 desalting column (GE Healthcare) or by repeated buffer exchange in sealed 10 kDa MWCO spin concentrators (Thermo Fisher) using an AccuSpin micro 17 centrifuge (Thermo Fisher). Samples incubated anaerobically with sulfite in sealed cuvettes were monitored by electronic absorbance until there were no further spectroscopic changes (~60 minutes) prior to MCD measurements. Electronic absorbance and magnetic circular dichroism (MCD): Absorption spectra were recorded using a model 4100 UV-visible-nIR spectrophotometer (Hitachi) or a model V-650 UV-visible spectrophotometer (JASCO). MCD spectra were recorded as described previously 3 with protein and buffer concentrations as shown in Table S1. Liquid Chromatography Mass Spectrometry (LC-MS): Samples were diluted 10-fold with 2% acetonitrile (MS grade, Honeywell), 0.1% formic acid (Fluka) in water (MS grade, VWR), transferred to LC-MS vials containing 250 μL inserts (Agilent), and sealed within the anaerobic chamber before removal. A 20 L aliquot of each sample was loaded onto a ProSwift RP-1S column (4.6 x 50 mm) (Thermo Scientific) on an Ultimate 3000 uHPLC system (Dionex, Leeds UK). Bound proteins were eluted (0.2 mL min −1 ) using a linear gradient over 15 min from 2% to 100% (v/v) acetonitrile, 0.1% (v/v) formic acid. The eluent was continuously infused into the electrospray ionization (ESI) source of a MicroTOF-QIII mass spectrometer, running Hystar (Bruker Daltonics, Coventry, UK) and operating in positive ion mode. The mass spectrometer was calibrated with ESI-L tuning mix (Agilent Technologies, California), which has m/z peaks in the range 118 - 2722. Compas Data Analysis 4.1, with Maximum Entropy v1.3, (Bruker) was used for processing of spectra under the LC peak, over mass range of 35 -40 kDa. Mass spectrometry data are presented as fractional abundance.
To record LC-MS of protein studied by protein film electrochemistry (see below), the protein coated mesoporous hierarchically structured indium-tin oxide (ITO) working electrode was removed from the electrochemical cell and covered with a drop of iodoacetate solution (100 mM) for 2 min. The entire ITO layer was then scraped into a solution of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid. The resulting suspension was then allowed to sediment by gravity (typically 5 min) so that the ITO particles settled. The protein containing solution was carefully recovered with a Hamilton syringe and analyzed as described above.
Protein Film Electrochemistry (PFE): Non-catalytic PFE was recorded using mesoporous hierarchically structured indium-tin oxide working electrodes 4 (20 µm thickness, 0.25 cm 2 surface area, 750 nm pore diameter) on fluoride-doped tin oxide coated glass, using the procedure detailed previously 3 except as follows. After reagent addition and buffer exchange in spin concentrators as described in Sample preparation, samples were drop cast onto ice-cold electrodes. Subtraction of baseline electrode responses from cyclic voltammograms was performed using NOVA 1.11 software to leave just protein (Faradaic) responses. The latter were fit to the theoretical Nernstian response for a surface-adsorbed species: where ΓO* is the surface population (mol cm −2 ) of adsorbed redox active species and Ae is the electrode area (cm 2 ). R, F and T have their usual meanings and the number of electrons transferred in the half-reaction (n) is 1. Reported Em values are averages obtained from the corresponding oxidative and reductive peak potentials. Catalytic PFE, e.g. Fig. 6A,B, was recorded using TsdA-coated, rotating, pyrolytic graphite edge working electrodes using the procedure detailed previously. 1 Aliquots of sulfite, tetrathionate and thiosulfate were introduced as required to produce the desired concentration in the electrochemical cell.
A) Cysligation of b-heme is retained by type-1 sites during the redox cycling associated with catalysis as illustrated here for cytochromes P450. B) Transitory Cysligation of b-heme is typical of type-2 sites for sensing heme and gas molecules. C) Cys -/His ligated c-heme. D) Sequence alignment for TsdAs from the indicated organisms. Heme 1 binding motif, proximal His ligand and distal Cys ligand are red on pink. Heme 2 binding motif, proximal His ligand and distal Met ligand are red on gray. Arg residues in the catalytic pocket of A. vinosum TsdA 5 are black on yellow.

RESULTS
LC-MS of CjTsdA proteins: To date, all LC-MS measured 6 for unmodified forms of CjTsdA expressed in Escherichia coli reports an additional mass of 99 ( 2) Da compared to that anticipated on the basis of the protein sequence with the addition of two hemes, an N-terminal Step II tag and four linker residues. 7 This is readily illustrated by the LC-MS for CjTsdA C138H (Fig. S2A) and CjTsdA C138M (Fig. S2B), which lack Cys 138 as a site of covalent modification. Both variants exhibit no significant heterogeneity and the observed (anticipated from plasmid sequence) masses are 37 240 (37 137) and 37 230 (37 131) Da for the C138M and C138H variants respectively. Experiments to date have unfortunately failed to identify the source of the additional mass of the protein but others have reported +98 adducts due to sulfate and/or phosphate bound non-covalently to peptides and proteins. 8 Accounting for this additional mass of the protein allows for straightforward interpretation of the LC-MS displayed by CjTsdA with cysteine modifications as shown in Fig. S3. Anticipated masses are shown in Fig. S4 for forms of TsdA that contain a modified Cys 138 . The LC-MS of as-prepared CjTsdA, Fig. S3A, without treatment with iodoacetate shows three-fold heterogeneity. One major species is observed near the mass associated with protein containing unmodified Cys 138 . A second, similarly abundant form, has a mass corresponding to the addition of either a single sulfur atom or two oxygen atoms, consistent respectively with persulfuration or sulfinylation of cysteine. A lack of high-spin species in electronic spectra of as-prepared TsdA 3 suggests that a sulfinylated form is not present in solution but does not rule out the possibility that oxidative modification occurs during handling and preparation of samples for LC-MS. A third, minor, species suggests a small population of cysteinyl thiosulfate.
S-6 Persulfurated and unmodified cysteine forms will differ from sulfinylated Cys 138 in that both will react with the alkylating agent iodoacetic acid and in the process be protected from adventitious oxidative modifications prior to LC-MS analysis. The LC-MS of TsdA treated with iodoacetic acid is shown in Fig. S3B. The major peak corresponds to the +57 Da shift that would result from alkylation of Cys 138 . Two smaller features lie at masses associated with alkylated persulfurated and cysteinyl thiosulfate. The absence of a feature near 37 234 Da implies that, although it contained the persulfurated form, there was no sulfinylated Cys 138 in the as-prepared TsdA.
To produce homogeneous Cys 138 -unmodified CjTsdA for the sulfite incubation experiments presented in the main text, samples were pre-treated with the disulfide reducing agent tris(2-carboxyethyl)phosphine 9-10 (TCEP). After TCEP treatment and subsequent trapping with iodoacetate the LC-MS, Fig. S3C and Fig. 2A, is dominated the acetylated cysteine form with some unreacted cysteine. TCEP treatment does therefore yield a homogeneous material with unmodified Cys 138 . Figure S4. Predicted masses (Da) of CjTsdA. The bracketed numbers in red show the mass changes for each species relative to the mass of CjTsdA with the post-translational (+99 Da) modification and an unmodified Cys 138 . All masses include the two covalently bound hemes. The nature of the +99 Da modification was not identified in this study but others have reported +98 adducts due to sulfate and/or phosphate bound non-covalently to peptides and proteins. 8